University of Connecticut OpenCommons@UConn Honors Scholar Theses Honors Scholar Program Spring 5-1-2014 Modeling the Adaptive Immune Response to Mutation-Generated Antigens Rory J. Geyer University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/srhonors_theses Part of the Allergy and Immunology Commons, Biochemistry Commons, Biological Engineering Commons, Biological Phenomena, Cell Phenomena, and Immunity Commons, Cancer Biology Commons, Disease Modeling Commons, Genetics Commons, Immunity Commons, Immunoprophylaxis and Therapy Commons, Medical Immunology Commons, and the Oncology Commons Recommended Citation Geyer, Rory J., "Modeling the Adaptive Immune Response to Mutation-Generated Antigens" (2014). Honors Scholar Theses. 392. https://opencommons.uconn.edu/srhonors_theses/392 Modeling the Adaptive Immune Response to Mutation-Generated Antigens Rory Geyer A Thesis Submitted in Partial Fulfillment of the Requirements for Graduation as an Honors Scholar and University Scholar at the University of Connecticut 2014 1 ABSTRACT Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional KnocKin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, while ovalbumin and CFP should only be expressed after a single base pair deletion that can restore the frame, has occurred. Experiments in vitro have been conducted to test whether the transgene behaves they way we expect. Transfection of EL4 cells with the transgene caused cells to express YFP. After growing the transfected cells (thus allowing time for spontaneous mutations), populations of YFP+CFP- and YFP+CFP+ cells were sorted and grown. The YFP+CFP- cells did not present SIINFEKL on H2-Kb on their surfaces, while YFP+CFP+ cells did; this was tested by incubating the cells with B3Z cells, which respond to the SIINFEKL/H2-Kb complex. The mouse model will be used to measure the rate of mutation in the transgene in vivo, investigate the immune response to mutation-generated antigens in a growing tumor in a live host, and test the hypothesis that an immune response to neoantigens on non- transformed somatic cells contributes to aging. 2 ACKNOWLEDGEMENTS Throughout my life, and especially throughout my last four years at UConn, I have been extremely fortunate in the people I have met and interacted with. These people have all played a part in my betterment as a student, a researcher, and a person and for that I owe them an incredible deal of thanKs. My parents have always supported my efforts in pursuing a career in academic medicine and have never told me to consider something that would be less difficult or offer more financial stability. I have always had their support in anything I wanted to pursue, and I continue to have it as I move on into the next phase of my life. I would like to thank my University Scholar project committee members, Drs. Adam Zweifach and Tony Vella for their insight into the early stages of the project and offering helpful suggestions toward its improvement. I would also liKe to thanK Dr. Zweifach for allowing me to get my start in research in his lab when I Knew absolutely nothing about research as a first semester freshman. The fours years I have spent in his lab have been valuable to me and I am grateful for all I have learned there. I owe much gratitude to all of the members of Dr. Srivastava’s lab for helpful conversations and suggestions, and for putting up with my inexperience. Fei Duan, Tatiana Shcheglova, AloK Das Mahapatra, Juliette Mouries, Adam Hagymasi, Nandini Acharya, and SuKrut KarandiKar were especially helpful with their advice and comments to improve both my project and myself as a researcher. They also helped by letting me borrow reagents, cells, and mice and taKing care of my cells and mice while I was at school. 3 I must thanK Dr. Basu for her constant help with planning experiments, analyzing data, tweaKing protocols, ordering mice and reagents, setting up new machines, and for allowing me to constantly nag her with any random question I had. Lastly, and most of all, I need to thanK Dr. Srivastava. I cannot come close to accurately expressing how grateful I am for all he has done for me over the past four years in this section. I developed my interest in cancer immunology in his lab and his guidance has offered me the absolute best preparation I could have received for a career in this field. He is my teacher, mentor, and friend and I cannot come close to described how privileged fortunate I am to have been his student and learn all that I have from him. 4 TABLE OF CONTENTS Title Page………………….………………………………………………………………………………………………1 Abstract………………………….…………………………………………………………….…………………..………2 AcKnowledgements………….…………………………………………………………….…………………...…..…3 Table of Contents…………….…………………………………………………………….…………………………..5 Introduction…………….…………………………………………………………….…………………….……………6 Random mutations accumulate with age and in tumor……………………………………..6 Anti-cancer immunity…………….…………………………………………………………….…………9 Thesis outline…………….…………………………………………………………….…………………...12 Materials and Methods…………….…………………………………………………………….………………...13 Results…………….…………………………………………………………….………………………………………..17 Creation of a cell line that expresses the construct….………………………………………17 Antigen-presenting ability of the transgene-expressing cell line….………………….19 Tissues of the transgenic mouse express the transgene….……………………………….20 Discussion…….…………………………………………………………….…………………………………………..21 Figures……….…………………………………………………………….……………………………………………..23 References……….…………………………………………………………….…………………………………..……29 5 INTRODUCTION RANDOM SOMATIC MUTATIONS ACCUMULATE WITH AGE AND IN TUMORS Random somatic mutations occur in all mitotic cells during the DNA synthesis phase of each passage through the cell cycle. Although there are DNA repair mechanisms in place to ensure fidelity in the synthesis of new DNA strands, these mechanisms do not worK with complete efficiency and some errors persist in the new strands (Echols and Goodman, 1991). Studies performed in cell lines have reported that random mutations occur at a rate between 10-9 and 10-5 mutations per base pair per cell division (BecKman and Loeb, 2005; Simpson, 1997). There are several types of mutations that occur during DNA synthesis. Among the most common are the insertion of an incorrect base leading to improper basepairing, the insertion of an entirely new base that was not previously present, and the deletion of a base (Zhang and Gerstein, 2003). Assuming they occur in a protein- coding gene, these different mutations can have vastly different effects on the structure (and thus the function) of the protein. Base pair substitutions may either be silent (not change the amino acid produced from the altered codon) or may cause a missense mutation (the amino acid produced from the altered codon is a different one than is produced from the unaltered codon). Silent mutations have no effect on the translated protein, but missense mutations may. Depending on how the new amino acid in the protein interacts with the others, the overall protein structure may or may not be changed. Insertion and deletions of bases tend to have a greater effect on the structure 6 of the mutated protein because they cause the nucleotides in the open reading frame to shift (hence, these are called frameshift mutations). This completely changes the codons that are read and, thus, the amino acids that are transcribed. Frameshift mutations tend to either lead to an entirely new protein structure or a truncated protein product from the generation of an early stop codon (Ogura et al., 2001). A particular type of insertion and deletion mutation is more liKely to occur in mononucleotide repeat tracts, which are 8 or more of the same nucleotide repeated consecutively (Stringer et al., 2004; see Levinson and Gutman, 1987). As DNA polymerase reads a mononucleotide repeat tract, a nucleotide in either the new strand or the template strand may “slip” bacKward (the template strand would move 3’ to 5’ and the new strand would move 5’ to 3’). Either strand may form a basepair with a nucleotide on the opposite strand that is upstream of the nucleotide it is supposed to pair with. If the template strand slips bacK, this will lead to a deletion in the new strand, whereas if the strand being synthesized slips back, it will have an insertion. Several trinucleotide repeat disorders are caused by slipped strand misparing mutations (Axford et al., 2013), including Huntington’s disease, in which CAG trinucleotide repeat tracts are expanded by insertions made in the newly synthesized strand (however, there is no frameshift with these diseases because three nucleotides “slip” at a time). Mutations (especially frameshift mutations) will often cause proteins to misfold and/or prevent them from functioning properly, which may lead to apoptosis of the cells harboring these mutations. However, in addition to causing various forms of cellular dysfunction, certain mutations drive tumorigenesis